Name: assays for the identification of novel antivirals against bluetongue virus
Uploaded: 2013-10-11T23:00:00
Description: Watch this Scientific Journal Video about Assays for the Identification of Novel Antivirals against Bluetongue Virus at JoVE.com

该项目由赠款1R03M​​H08127-01和7R03MH08127-02由美国国立卫生研究院对问李的支持，并通过从医学系在UAB的资金影响到Q力。从Molette基金和奥本大学的支持表示赞赏。我们也感谢工作过程中从普林车女士和沃洛Musiienko先生的技术协助。

1。细胞，病毒和抗病毒化合物<ol><li>维护的BSR细胞中，幼仓鼠肾（BHK）细胞10，在Dulbecco改良的Eagle培养基（DMEM）含有5％胎牛血清（FCS）的衍生物，100U/ml青霉素和100微克/毫升链霉素。 </li><li>对于所有三个实验，板细胞的DMEM含1％FCS，100U/ml青霉素和100微克/毫升链霉素，为优化以前5。该培养基被称为对于所有三个实验测定培养基。 </li><li>孵育中所有单元的培养箱中在37℃，含5％C...

<ol>
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Virol. 78, 531-538 (2004).</li><li>Puig-Basagoiti, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assays+using+a+luciferase-expressing+replicon,+virus-like+particles,+and+full-length+virus+for+West+Nile+virus+drug+discovery.">High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery.</a> Antimicrobial Agents and Chemotherapy. 49, 4980-4988 (2005).</li><li>Puig-Basagoiti, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triaryl+pyrazoline+compound+inhibits+flavivirus+RNA+replication.">Triaryl pyrazoline compound inhibits flavivirus RNA replication.</a> Antimicrob. Agents Chemother. 50, 1320-1329 (2006).</li><li>Duan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+protection+against+the+highly+pathogenic+H5N1+influenza+virus+by+an+antisense+phosphorothioate+oligonucleotide.">In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide.</a> Antivir. Ther. 13, 109-114 (2008).</li><li>Ray, D., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+flavivirus+antiviral+drug+discovery+and+vaccine+development.">Recent advances in flavivirus antiviral drug discovery and vaccine development.</a> Recent Pat. Antiinfect. Drug Discov. 1, 45-55 (2006).</li><li>Severson, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+a+100,000-compound+library+for+inhibitors+of+influenza+A+virus+(H3N2).">High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2).</a> J. Biomol. Screen. 13, 879-887 (2008).</li><li>Severson, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+high-throughput+screen+for+inhibitors+of+SARS+CoV+and+its+application+in+screening+of+a+100,000-compound+library.">Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library.</a> J. Biomol. Screen. 12, 33-40 (2007).</li><li>Bolken, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+potent+small+molecule+inhibitor+of+hemorrhagic+fever+New+World+arenaviruses.">Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.</a> Antivir. Res. 69, 86-97 (2006).</li><li>Van Loock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+cellular+screening+assay+for+the+discovery+of+HIV-1+integrase+inhibitors.">A novel high-throughput cellular screening assay for the discovery of HIV-1 integrase inhibitors.</a> J. Virol. 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抗病毒的命中初始识别，用于抗病毒药物发现和开发的关键步骤之一是开发强大的测定法，其中包括选择一个量化指标，开发一种简单的协议，从而获得足够的信号和低于10％的CV。大多数生化或基于细胞的筛选被设计成由于在筛分过程所需的再现性和潜在的大量分子筛选的提供基于最稳健的，简单和便宜的测定法，化学的起点。该CPE为基础的检测被指定为满足这些要求，以确定从一个大的复合图...

BTV是一个原型双链RNA病毒属中的环状病毒 ， 呼肠弧病毒家族。 BTV是家畜中最重要的疾病，包括绵羊，山羊，牛等家畜之一，与世界各地的1,2 $ 3十亿/年亏损。异国情调的BTV血清型是在上市的重要动物病原体“美国农业部的严重后果家畜病原体。”重新出现的BTV的最近，已经引起疾病的牛大爆发和羊在整个北欧3,4几个国家。作为其经济意义的结果，作为一个模型系统，BTV已经成为广泛的分子，遗传和结构研究的主题，并且几种疫苗已经开发出来。然而，由于缺乏适当的检测对抗病毒药物的发现，有没有可用的对抗BTV抗病毒药物。 在最近的高通量筛选（HTS）活动使用BTV作为模型系统，我们ðeveloped，优化和验证一个基于CPE的检测，以确定对虫媒病毒5潜在的广谱抗病毒药物。基于CPE的测定是，已在抗病毒的药物发现对许多病毒诱导快速和可观察到的CPE /凋亡5-7被用于一个公认的测定。在我们的系统，北京电视台后感染，CPE是显而易见的脊椎动物细胞，包括海拉，BSR和HEK 293T 8。 BTV-诱 ​​导的CPE可以监测和量化使用各种细胞活力的检测方法，包括的CellTiter格洛细胞活力试剂盒（试剂盒CTG）9。该试剂盒确定基于对细胞内ATP的定量在培养的活细胞的数目呈现，这标志着代谢活跃的活细胞的存在。在优化条件下，这里提出的基于CPE的检测表明其可行性与“混合和计量”一步到位的协议，并与稳定的荧光信号的灵活性。同时，有毒化合物热度庆安细胞活力将被排除在这个基于CPE的检测。在基于CPE的检测表明其稳定性和抗病毒的药物发现对抗BTV的可靠性，并且已经用于筛选NIH的分子库的小分子库（MLSMR），从而导致潜在的抗病毒铅化合物（次六个新集群的识别）5。 当一个潜在的抗病毒化合物中已使用基于CPE测定法确定，这将需要经受10浓度的剂量-反应实验来确定抗病毒活性和细胞毒性2的范围内。的抗病毒效力，表示为50％抑制浓度（IC 50）或50％有效浓度（EC 50），是一种药物，其抑制基线和最大之间病毒诱导的CPE的中途的浓度。抗病毒药物的细胞毒性， 即 50％的细胞毒性浓度（CC 50），是浓度的药物诱导的基线值和最大值之间的细胞毒性的50％。选择性指数（SI），记为50％的SI（SI 50）从CC 50 / IC 50，它确定对病毒诱导的CPE抗病毒的特异性来计算。该IC 50（或EC 50），CC 50和SI 50值是至关重要的措施，以确定抗病毒化合物是否是有效的和有选择性的进行进一步的药物开发。 当一种抗病毒药表明没有明显的毒性在体外 ，还防止了病毒诱导的CPE和生产性病毒的生命周期，它以表征其农业部2是重要的。我们通过开展ToA的检测，以确定病毒的生命周期是受抗病毒的可能（S）步实施这种表征。通常，抗病毒化合物加入到细胞中在不同的时间前或后病毒感染。如果抗病毒药物加入到被感染的细胞中张贴到其目标STEP感染的过程中，它比较时向其中添加之前的步骤中的人会导致较低的活性。因此，ToA的研究是一种用于确定化合物的抗病毒效力，其潜在目标的关键，无论是在病毒的生命周期或参与病毒生命周期的宿主机器。 对于所有三个实验，细胞存活率用CTG套件下列制造商的指示5确定。该检测系统输出，可以使用不同的内部软件进行分析，充分的发光信号。每个测定的至少一式三份用8副本进行验证和执行。对于所有获得的数据，三个参数，包括平均值（AVE），标准偏差（STDEV），和共同有效的变化（CV）进行分析，以确定检测的鲁棒性。一次测定的鲁棒性进行了测定，将数据进行进一步的分析，以及使用各种生物统计学和图形化绘制C工具2。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>1134218</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>1000185</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gbico</td>
			<td>1049769</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>CellTiter-Glo (CTG) kit</td>
			<td>Promega</td>
			<td>TB288</td>
			<td>For cell viability measurement</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>S25309B</td>
			<td>Diluted from 95%</td>
		</tr>
		<tr>
			<td>Antiviral huashilcompounds</td>
			<td>NIH MLSMR and de novo synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BTV-10</td>
			<td>ATCC</td>
			<td>VR-187</td>
			<td></td>
		</tr>
		<tr>
			<td>BSR cell</td>
			<td>Developed in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy-II multi-mode microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td>For luminescent signal reading</td>
		</tr>
		<tr>
			<td>MicroFlo select dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td>Adding cells, virus, and reagents</td>
		</tr>
		<tr>
			<td>384-well flat-bottom microplate</td>
			<td>CORNING</td>
			<td>28908031</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gen. 5 software</td>
			<td>BioTek</td>
			<td></td>
			<td>For analysis of reading outputs from Synergy-II multi-mode microplate reader</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>Version 5</td>
			<td>For biostatic analysis and plot</td>
		</tr>
	</tbody>
</table>

assays for the identification of novel antivirals against bluetongue virus

为了找出对BTV潜在的抗病毒药物，我们已经开发，优化和验证这里提出三个实验。在基于CPE测定法的开发是为了评估在第一测定化合物是否表现出任何抗病毒活性，并已用于筛选大量化合物库。同时，抗病毒药物的毒性也可以使用基于CPE的测定法进行评价。剂量反应实验的目的是确定疗效所选抗病毒的范围内， 即 50％抑制浓度（IC 50）或有效浓度（EC 50），以及它的范围的细胞毒性（CC 50）。 TOA的分析受聘为农业部初步研究，以确定在BTV病毒的生命周期或宿主细胞机制的可能影响本小说抗病毒药物的作用机制。这些测定是在细胞培养系统的抗病毒功效的评价是至关重要的，并已被用于我们最近的研究导致ing到了一些对BTV新的抗病毒药物的鉴定。

1。化合物的抗病毒疗效基于细胞的CPE测定法的开发，优化和使用该发光基CTG试剂盒，以确定新的抗病毒药物对抗BTV如先前描述的2,5验证体外 。在10剂量响应测定法来反映抗病毒疗效通过测量基础上，在活细胞中5,11呈现细胞ATP的定量在培养代谢活细胞的数目的确定铅化合物和细胞毒性。在我们以前的报告中，一些潜在的抗病毒化合物进行了评价，包括通?...

Watch this Scientific Journal Video about Assays for the Identification of Novel Antivirals against Bluetongue Virus at JoVE.com

Assays for the Identification of Novel Antivirals against Bluetongue Virus

三个实验，包括细胞病变效应（CPE）为基础的试验中，剂量反应实验和时间的 - 加法（TOA）测定法已被开发，优化，验证和用于识别对蓝舌病毒的新的抗病毒药物（BTV），以及以确定可能的机制， - 行动（MOA）为新发现的抗病毒药物。

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Research

JoVE Journal

Immunology and Infection

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

病毒浓度的测定通过空斑试验：用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

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Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...